Abstract Genetic dissection of Cardiac Conduction System homeostasis and regeneration The cardiac conduction system (CCS) is required for initiating and maintaining regular rhythmic heartbeats. CCS defects commonly give rise to arrhythmia, a leading cause of morbidity and death worldwide. CCS dysfunction can be inherited or acquired due to conditions such as drug toxicity or myocardial infarction. It is imperative to elucidate the molecular mechanisms underlying CCS homeostasis to facilitate development of cardiac therapies. Importantly, these mechanisms are poorly understood owing to numerous technical challenges. Hippo signaling, a pivotal organ size control pathway, inhibits cardiomyocyte proliferation and regeneration. However, the role of Hippo signaling in the CCS is unclear. Here we will determine whether Hippo signaling regulates CCS homeostasis. Additionally, we will identify regulators and targets of Hippo signaling in the CCS. Our preliminary observations revealed that disruption of Hippo signaling in the CCS caused cardiac arrhythmias in mice, suggesting an important role of Hippo signaling in CCS homeostasis. Notably, deletion of Hippo signaling rescued cardiac rhythm and function after CCS cell ablation, suggesting that repression of Hippo signaling protects the CCS from damage. In addition, we identified candidate microRNA regulators and downstream targets of Hippo signaling. Here we propose to investigate the function and molecular regulatory mechanism of Hippo signaling in the CCS through in the following specific aims: 1) Define the role of Hippo signaling in CCS homeostasis and elucidate the mechanism by which repression of Hippo signaling protects the CCS from damage, 2) Identify upstream microRNA regulators of Hippo signaling in the CCS, and 3) Identify downstream targets of the Hippo pathway that modulate CCS function.